Re: Growth-inhibitory effect of heparin on Babesia parasites.

[Guest guest]

thanx nelly. that means i have to know what the optimal dose is, as

they were increased by low concentrations?

i wish i never walked in that garden 5 years ago. its just too

overwhelming right now.

-- In infections , " Nelly Pointis "

<janel@p...> wrote:

> Jill,

> You might want to check this out

> Nelly

>

> Antimicrob Agents Chemother. 2004 Jan;48(1):236-41. Related

Articles, Links

>

>

> Growth-inhibitory effect of heparin on Babesia parasites.

>

> Bork S, Yokoyama N, Ikehara Y, Kumar S, Sugimoto C, Igarashi I.

>

> National Research Center for Protozoan Diseases, Obihiro University

of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido

080-8555, Japan.

>

> We examined the inhibitory effects of three heparins on the growth

of Babesia parasites. The multiplication of Babesia bovis, B.

bigemina, B. equi, and B. caballi in in vitro cultures and that of B.

microti in vivo were significantly inhibited in the presence of

heparins, as determined by light microscopy. Treatment with various

concentrations of heparin showed complete clearance of the

intracellular parasites. Interestingly, a higher percentage of

abnormally multidividing B. bovis parasites was observed in the

presence of low concentrations of heparin. Furthermore, fluorescein

isothiocyanate-labeled heparin was preferably found on the surfaces

of extracellular merozoites, as detected by confocal laser scanning

microscopy. These findings indicate that the heparin covers the

surfaces of babesial merozoites and inhibits their subsequent

invasion of erythrocytes.

>

> PMID: 14693545 [PubMed - indexed for MEDLINE]

[Guest guest]

Jill,

Did you read the whole article? They go into a lot more details re doses.

Nelly

(quote from the full text)

In vivo effect of heparin against B. microti infection. Having observed that heparin 1 had a strong inhibitory effect on the growth of the cultured parasites, we were encouraged to investigate its in vivo effect on B. microti infection in mice (Fig. 5A). When the infected mice showed about 1% parasitemia, they received subcutaneous injections of either 500, 250, 100, 20, or 4 U of heparin for 10 consecutive days. In the treated group, the levels of parasitemia increased significantly more slowly, achieving peak parasitemias of 10% (in the presence of 500 U), 15% (250 U), 17% (100 U), and 34% (20 U) on day 8 after inoculation. In contrast, in the control group, the peak parasitemia of more than 72% occurred on day 7 after inoculation. On the basis of these peak parasitemias, the IC50 for B. microti infection was calculated to be 16 U (81 µg) per mouse. In several mice treated with 250 and 500 U of heparin, hematomas occurred around the puncture site following the injections, and among the 10 mice in each of the groups treated with 500 and 250 U of heparin, three and one mice died, respectively, following the second or third injection. As for the other Babesia parasites, a greater percentage of free merozoites was seen in the heparin-treated groups, in which the peak proportion of free merozoites of 45% occurred on day 5 in the group treated with 100 U of heparin, while the highest value did not exceed 32% in the control group (data not shown). Additionally, binding of heparin-FITC was also observed on the surfaces of free merozoites (Fig. 5B). There were no significant differences in the percentages of the Maltese cross forms among the groups (data not shown).

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FIG. 5. (A) Inhibitory effects of 500, 250, 100, 20, or 4 U of heparin 1 per mouse on the course of B. microti infection in mice. Each value represents the mean ± standard deviation for observations for 10 mice in each group. *, statistically significant differences (P < 0.01) between the group treated with 100 U of heparin and the control group; #, times of subcutaneous inoculation of heparin or the control reagent (PBS). ( Localization of heparin-recognizing locus. B. microti-infected blood was preincubated with heparin-FITC, followed by fixation with acetone-methanol, and was then examined under a confocal laser scanning microscope. The heparin-antigen reaction (green) and nucleus (red) were visualized by staining with FITC and PI, respectively. Distinct fluorescence was detectable around free merozoites (arrow) but not the infected RBC. Bar, 5 µm.

DISCUSSION In the present study, different kinds of heparins were found to significantly inhibit the growth of Babesia parasites. The increase in the percentage of free merozoites of all parasites tested and the occurrence of an abnormally high percentage of the multidividing trophozoite form of B. bovis might be due to the blocking effects of heparin on the invasion of merozoites into RBCs and on the release of mature trophozoites from infected RBCs. In the presence of a low concentration of heparin, some B. bovis merozoites still retained their invasive capacity and subsequently divided in the infected RBCs. However, we conclude that the heparin-exposed parasites are not able to escape from the infected RBCs, probably because they have been carried over into the erythrocytic cytoplasm, resulting in an abnormal progression to further trophozoite division in the RBCs. Functionally, the multidividing form of B. bovis is not related to the Maltese cross form observed only in B. equi and B. microti because the proportion of the Maltese cross form of these parasites never increased, even in the presence of heparin.

The study with heparin-FITC indicated the presence of a molecule(s) with an affinity for heparin on the surfaces of free merozoites. Because heparin at a high concentration completely inhibited the invasion of merozoites, the interaction between the molecule with an affinity for heparin and exogenous heparin must play a critical role in disruption of the invasion process. Previous studies with heparin-FITC-labeled Toxoplasma gondii found that the fluorescence is localized near the subapical region of free tachyzoites (6). T. gondii surface antigen (SAG3) is known to show a heparin-binding property and mediates the attachment of the tachyzoite to the cellular HS proteoglycan of host cells (22). In P. falciparum, heparin interacts with the circumsporozoite protein expressed on the surface of the sporozoite (34). The binding of the circumsporozoite protein to cellular HS proteoglycans is required for sporozoite attachment to hepatocytes (33). The molecule(s) with an affinity for heparin observed in this study might also be essential for the process of invasion of RBCs by Babesia parasites. In several reports (40, 44), the HS-like GAG was described to be located on the surfaces of RBCs, suggesting the possibility that the molecule(s) of the free merozoite with an affinity for heparin might recognize the HS-like GAG as the binding partner, similar to the HS used for recognition by some other parasitic microbes (35).

In the erythrocytic stage of P. falciparum, heparin-FITC is detectable on the surfaces of infected RBCs (3). The infected RBCs, including the mature trophozoites, are bound deep in the microvasculature, primarily to endothelial cells (cytoadherence) and also to normal RBCs (rosetting) (8, 12, 15, 42). The sequestration is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed as a parasite-derived polypeptide on the surfaces of infected RBCs (43) and which adheres to HS on endothelial cells and also to the HS-like GAG on normal RBCs (3). The resultant binding of infected RBCs in the inner organs prevents the parasite from being removed by splenic clearance (10, 37). Although it has also been suggested that some Babesia parasites sequester infected RBCs in the host microvasculature (1), the mechanism has not been fully determined.

In this study, the IC50 of heparin for the rodent babesia isolates was determined to be 16 U (81 µg) per mouse. *******However, some side effects (the occurrence of hematomas and the death of a total of four mice) were observed during treatment with either 500 or 250 U of heparin. These adverse reactions might be caused by the anticoagulation effect of heparin as a result of heparin binding to antithrombin AT-III and subsequent inhibition of the blood coagulation cascade (23, 41). *******Therefore, for babesiacidal therapy the application of heparin doses as high as those mentioned above is not recommended, even though these doses suppress parasite growth. *******Since we used unfractionated forms of heparin preparations in our study, it remains unknown which fraction of heparin exerts the highest degree of efficacy in the suppression of Babesia parasites without causing side effects. Further studies will be required to develop safety measures for heparin as therapy for babesiosis.

The present results suggest that the Babesia parasites do not have the PfEMP1-like molecule that confers a heparin-binding property to the surfaces of infected RBCs. However, the surfaces of extracellular merozoites of equine, bovine, and rodent Babesia parasites express the molecule(s) with an affinity for heparin. Furthermore, their covering with heparin was suggested to interfere with subsequent invasion of the RBCs. It might also be possible that the HS-like GAG on RBCs acts as a host receptor for merozoite attachment. There has been no report of the presence of a molecule with an affinity for heparin on the surfaces of free merozoites of Plasmodium parasites. Further investigation of the growth inhibition caused by heparin will be helpful in providing an understanding of the process of merozoite invasion. In particular, it will be very important to identify the molecule(s) with an affinity for heparin.

[infections] Re: Growth-inhibitory effect of heparin on Babesia parasites.

thanx nelly. that means i have to know what the optimal dose is, as they were increased by low concentrations?i wish i never walked in that garden 5 years ago. its just too overwhelming right now.-- In infections , "Nelly Pointis" <janel@p...> wrote:> Jill, > You might want to check this out > Nelly> > Antimicrob Agents Chemother. 2004 Jan;48(1):236-41. Related Articles, Links > > > Growth-inhibitory effect of heparin on Babesia parasites.> > Bork S, Yokoyama N, Ikehara Y, Kumar S, Sugimoto C, Igarashi I.> > National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.> > We examined the inhibitory effects of three heparins on the growth of Babesia parasites. The multiplication of Babesia bovis, B. bigemina, B. equi, and B. caballi in in vitro cultures and that of B. microti in vivo were significantly inhibited in the presence of heparins, as determined by light microscopy. Treatment with various concentrations of heparin showed complete clearance of the intracellular parasites. Interestingly, a higher percentage of abnormally multidividing B. bovis parasites was observed in the presence of low concentrations of heparin. Furthermore, fluorescein isothiocyanate-labeled heparin was preferably found on the surfaces of extracellular merozoites, as detected by confocal laser scanning microscopy. These findings indicate that the heparin covers the surfaces of babesial merozoites and inhibits their subsequent invasion of erythrocytes.> > PMID: 14693545 [PubMed - indexed for MEDLINE]

[Guest guest]

Jelly,

Were you and/or daughter diagnosed with any genetic coagulation defects that could explain the hypercoagulation you suffered or do you attribute it mainly to infection/inflammation?

Soory, you might've told us already, I don't always read everything that gets posted on the list.

Nelly

[infections] Re: Growth-inhibitory effect of heparin on Babesia parasites.

This is good Nelly. I hadn't found the whole study. Thanks!> Jill,> > Did you read the whole article? They go into a lot more details re doses.> > Nelly> > (quote from the full text)>

[Guest guest]

I was tested for all of the genetic stuff and came back with

nothing. I was sure I was going to have Factor V Liden, but nope. My

coagulation problems were determined to then be due to chronic

infection. My daughter wasn't geneticly tested, but after the 3

years of heparin, I guess she was more like 2 1/2 years, her

coagulation " appears " to be normal. At least she doesn't have the

ice cold hands and feet that were usually blue. It was so bad for

her that her lips sometimes turned blue.

We still aren't entirely out of the woods though. I can still get a

pretty serious herx if I take ABX. She still has some bad days, but

is raising two kids and working 2 nights and 2 days. She is very

phsycially active.

So we just keep plugging along. I use the NattoKinase now instead of

the heparin. It seems to be a real good replacement. I was testd

last Oct. with the ISAC panel and my coagulation looked REAL GOOD.

Both of us will be starting ABX in the near future to wipe out any

residule that might be left.

> > Jill,

> >

> > Did you read the whole article? They go into a lot more

details re

> doses.

> >

> > Nelly

> >

> > (quote from the full text)

> >